Towards a ‘clicked’ PSMA targeting gene delivery bioconjugate-polyplex for prostate cancer

Prostate cancer is the most common cancer in men in the UK with over 50 000 new cases diagnosed each year and although therapeutic advances in surgery, anti-androgens, radio- and chemotherapy have increased survival rates, there still remains a need for new treatments to combat the most aggressive forms of the disease. Gene therapy offers promise as an alternative approach but is reliant on selective targeting to the cancer cell surface. Herein we describe the novel construction of a prostate specific membrane antigen (PSMA) binding bioconjugate-polyplex, based on a glutamate–urea peptide scaffold using ‘click’ chemistry, which we demonstrate is capable of targeted delivery of a GFP gene to PSMA overexpressing prostate cancer cells, and therefore may have potential future application as part of a prostate cancer gene delivery therapy.


Synthesis of the linear anti-PSMA peptide S4
Solid phase peptide synthesis (SPPS) was used for making the resin-bound short peptide, adopted from Eder et al 1 .
To 250 mg (0.305 mmol) of 2-chlorotrityl resin (swollen in dry DCM) was added 276 mg (0.61 mmol) of Fmoc-Lys(Alloc)-OH and 266 µL (1.53 mmol) of DIPEA (both dissolved in 3 mL of dry DCM).The mixture was left stirring at room temperature for 2 hours.The solution was then filtered out and the resin was washed two times with dry DCM followed by two washes (10 min each) with the capping solution DCM:MeOH:DIPEA (18:1.5:1.5 v/v).The resin was washed successively with DMF (5 x 2min), DCM, and MeOH (3 x 2min each) and dried under vacuum.The loading capacity of the resin was then quantified and found to be 0.67 mmol.g -1 .
The Fmoc group was then cleaved off by swelling the resin in DMF for 30 min and washing with 20% piperidine in DMF (5 x 2min).The resin was then washed successively with DMF (5 x 2min), DCM, and MeOH (3 x 2min each) and dried under vacuum.
For synthesising the glutamyl residue, the isocyanate species was generated in situ as follows: to an ice-cold solution of triphosgene (188 mg, 0.633 mmol) in 5 mL dry DCM was slowly added a mixture of H-Glu(OtBu)-OtBu (562 mg, 1.9 mmol) and DIPEA (1 ml) in 30 mL of dry DCM.The mixture was left stirring on ice for an hour followed by 2 hours at room temperature.The resin (315 mg, equating to 0.211 mmol of Fmoc-Lys(Alloc)-OH) was then added to the solution which was gently stirred overnight.The solution was then filtered out and the Alloc group was cleaved off by agitating the resin with tetrakis(triphenylphosphine)palladium(0) (61 mg, 0.053 mmol) and morpholine (469 µL, 5.3 mmol) in DCM.The resin was then washed with DCM (5 x 2 min) then MeOH (3 x 2 min) and dried under vacuum.The rest of the anti-PSMA moiety was synthesised using standard Fmoc chemistry: Fmoc-3-(2-Naphthyl)-L-alanine (461 mg, 1.05 mmol), HCTU (436 mg, 1.05 mmol), and DIPEA (404 µL, 2.32 mmol) were dissolved in 3 mL of DMF and the solution added to the resin.This mixture was gently agitated for 1 hour, then the resin was filtered and washed with DMF (3 x 2min).Fmoc deprotection was achieved by agitating the resin with a solution of 20% piperidine in DMF (5 x 2min).The resin was washed with DMF (5 x 2min) prior to the next coupling.The above steps were repeated for the addition of Fmoc-tranexamic acid (399 mg, 1.05 mmol).
A test cleavage was carried out on S3 after addition of the tranexamic acid, with characterisation by HPLC-MS.The rest of the linear peptide S4 was synthesised as above using the following order of the amino acids (1.05 mmol each): Fmoc-Glycine, Fmoc-Serine, Fmoc-Glycine, Fmoc-Serine, Fmoc-Lysine (Alloc), Fmoc-Serine, Fmoc-Glycine, Fmoc-Serine, Fmoc-Glycine.
The resin was finally washed with DCM, and MeOH (3 x 2min each) and was then dried under vacuum.

Synthesis of fluorescent anti-PSMA peptide 1
115 mg (0.077 mmol) of the linear anti-PSMA peptide resin S4 was swollen in DMF for 30 min.Boc-Ser(tBu)-OH (101 mg, 0.385 mmol), HCTU (160 mg, 0.385 mmol), and DIPEA (148 µL, 0.847 mmol) were dissolved in 3 mL of DMF and added to the resin which was gently agitated for 1 hour.The solvent was then removed and the resin was washed with DMF (5 x 2min) and DCM (3 x 2min).The Alloc group was cleaved off by gently agitating the resin with tetrakis(triphenylphosphine)palladium(0) (22 mg, 0.019 mmol) and morpholine (170 µL, 1.92 mmol) in DCM for 1h.The resin was then washed with DCM and DMF (5 x 2min each) and filtered.60 mg (0.154 mmol) of fluorescein isothiocyanate isomer I and 148 µL (0.847 mmol) of DIPEA was dissolved in 3 mL of DMF and added to the resin.The mixture was gently agitated in the dark overnight.The resin was then washed with DMF (5 x 2min) followed by DCM and MeOH (3 x 2min each) and was dried under vacuum overnight.
The target peptide was then cleaved off the resin by gently agitating the resin with a mixture of 95:2.5:2.5 (v/v) of TFA:H2O:triisopropylsilane for 1 hour.The solution was added to ice-cold Et2O to precipitate the peptide which was then centrifuged at 4000rpm 4 o C for 10 min to give a pellet.The pellet was washed with cold Et2O and centrifuged 3 times, then dissolved in 10% aq.acetic acid and freeze-dried overnight.LNCaP and PC3 cells were detached using 10 mM EDTA in PBS, washed, re-suspended in ice cold PBS and plated in a 96 well plate at 75, 000 cells per well.Cells were incubated for 1.5 hour at 4 °C with 1 at serial dilutions (1:3) of 50 g/ml -2.9x10 -8 μg mL −1 .Cells were then washed, fixed overnight in FACS buffer (PBS 1% FCS, 0.05% sodium azide) containing 1% formaldehyde and resuspended in FACS buffer for analysis on a Cytoflex S (Beckmann Coulter).A Live/Dead stain was used on cells only for gating purposes.Median fluorescent units of cells only were subtracted from cells plus peptide giving specific fluorescent units.Data was analysed using GraphPad Prism, One site -Specific binding.The Kd was 0.1663 ng/ml, 0.085 nM.
The resin was then washed with DMF (5 x 2min) and DCM (3 x 2min).The Alloc group was cleaved off by gently agitating the resin with tetrakis(triphenylphosphine)palladium(0) (44 mg, 0.038 mmol) and phenylsilane (468 l, 3.8 mmol) in DCM for 1h.The resin was then washed with DCM and DMF (5 x 2min each) and filtered.The resin was shrunk by washing with DCM (3x 2min) and MeOH (3x 2min) with rotation.The resin was dried initially on a vacuum manifold and then on high vacuum line overnight.
Roughly one third of this resin was taken and swollen in DMF for 30 min.Fmoc-PEG2-OH (131 mg, 0.234 mmol), HCTU (97 mg, 0.233 mmol) and DIPEA (95 l, 0.515 mmol) were dissolved in 1 ml DMF and added to the resin which was gently agitated for 2 hours.The resin was then washed with DMF (5 x 2min) followed by DCM and MeOH (3x 2min each) and was dried under vacuum overnight.
The target peptide was then cleaved off the resin by gently agitating the resin with a mixture of 95:2.5:2.5 (v/v) of TFA:H2O:triisopropylsilane for 1 hour.The solution was added to ice-cold Et2O to precipitate the peptide which was then centrifuged at 4000 rpm 4 o C for 10 min to give a pellet.The pellet was washed with cold Et2O and centrifuged 3 times, then dissolved in 10% aq.acetic acid and freeze-dried overnight affording a colourless solid S10.HRMS-ESI: found [M+H+Na] 2+ 915.4205,C78H123N18NaO31 2+ requires 915.4244.

Synthesis of anti-PSMA DBCO peptide 3
To 583 l of a 3.3 mM solution of anti-PSMA PEG peptide S10 in anhydrous DMF was added 3 equivalents of DBCO NHS ester (Sigma 761524) (159 l of a 50 mM stock in DMF) and 1 l of triethylamine.The resultant solution was stirred at room temperature overnight after which time it was diluted with water and lyophilised The lyophilised material was re-suspended in 5% DMF/water (2 ml), centrifuged and the supernatant was applied to a C18 column prewashed with methanol and then water.The column was eluted with a water/acetonitrile gradient.Fractions were checked by LCMS (Figure S8), the product 3 eluted in 40% acetonitrile/water and lyophilised to afford a colourless solid.

Calculation of the number of azides on PEI25K-g(20)-PEG3.4K-N3
A series of DMSO solutions were prepared such that the final volumes were 1 mL and the quantities of azide functionalised polymer 2 (PEI25K-g(20)-PEG3.4K-N3,Nanosoft Polymers) and DBCO-acid added to each solution were known.This enabled the concentrations of each of these species in the solution to be calculated.One series of solutions contained both DBCO-acid and polymer (see Table S1 below), whereas the other series lacked polymer but contained akin concentrations of DBCO-acid (see Table S2 below).The solutions were incubated overnight at rt in darkness, after which time the UV-vis spectra of the solutions were recorded.
Unreacted DBCO absorbs strongly at 313 nm, whereas the triazole product of the reaction of DBCO with an azide does not.By comparing the absorbance at 313 nm between comparable solutions in the two series, it is therefore possible to obtain a value for ∆   .This value could then be used to determine much DBCO-acid has been consumed in each "Polymer and DBCO-acid" solution using the Beer-Lambert law  = , where      was determined experimentally to be 14000 dm 3 mol -1 cm -1 , and  was 1 cm.

∆𝑨𝒃𝒔 𝟑𝟏𝟑 𝒏𝒎 = (𝑨𝒃𝒔 𝟑𝟏𝟑 𝒏𝒎 𝑷𝒐𝒍𝒚𝒎𝒆𝒓 𝒂𝒏𝒅 𝑫𝑩𝑪𝑶 𝒂𝒄𝒊𝒅) − (𝑨𝒃𝒔 𝟑𝟏𝟑 𝒏𝒎 𝑫𝑩𝑪𝑶 𝒂𝒄𝒊𝒅 𝒐𝒏𝒍𝒚) [𝑫𝑩𝑪𝑶 𝒄𝒐𝒏𝒔𝒖𝒎𝒆𝒅] = − ∆𝑨𝒃𝒔 𝟑𝟏𝟑 𝒏𝒎 𝜺 𝑫𝑩𝑪𝑶 𝒂𝒄𝒊𝒅 𝒂𝒕 𝟑𝟏𝟑𝒏𝒎
We define the equivalents of DBCO delivered to the "Polymer and DBCO-acid" solutions as and we define the equivalents of DBCO consumed per polymer chain as Ideally, the maximum equivalents of DBCO that could be consumed per polymer chain would be expected to equate to the quantity of azides present on each polymer chain.After DBCO is delivered to the polymer solutions and the resultant solutions are incubated for a fixed time period, the equivalents of DBCO consumed will therefore tend towards the number of azides present on each polymer chain.This behaviour will take the form of an exponential function of the equivalents of DBCO that were delivered to the "Polymer and DBCO-acid" solutions: = ( −  −(   ) ) Where A = the number of azides available for reaction per polymer chain, and β is an undefined preexponential factor.
Non-linear regression of a plot of     vs     can therefore be used to determine the value of A, and by extension, the number of azides available for reaction per polymer chain.

Synthesis of anti-PSMA polymer 4
A 1 mM solution of 3 in DMSO was prepared alongside a 20 M solution of PEI25K-g(20)-PEG3.4K-N3 2 (Nanosoft Polymers) in DMSO. 100 µL reactions were set up as detailed in Table S3 below and incubated at room temperature overnight.The following day reactions were diluted with water to less than 10% DMSO and dialysed into water before freeze drying.Reactions were monitored by UV-vis (Figure 4B).

A
final purification step was performed by dissolving the peptide in LC-MS grade water and loading it on a C18 solid-phase extraction (SPE) cartridge.The target peptide was eluted with 40% MeCN in water.The solution was lyophilised to give a bright yellow residue 1 (114 mg, 80%).HRMS-ESI: Found [M+2Na] 2+ 940.8344,C83H105N17Na2O29S 2+ requires 940.8385.

Table S1 :
The concentrations of species present in samples of Polymer and DBCO-acid in DMSO, and the absorbance of these solutions at 313 nm.

Table S2 :
The concentrations of samples of DBCO-acid in DMSO and their absorbance values at 313 nm.